The transporter associated with antigen processing: function and implications in human diseases.

The adaptive immune systems have evolved to protect the organism against pathogens encountering the host. Extracellular occurring viruses or bacteria are mainly bound by antibodies from the humoral branch of the immune response, whereas infected or malignant cells are identified and eliminated by the cellular immune system. To enable the recognition, proteins are cleaved into peptides in the cytosol and are presented on the cell surface by class I molecules of the major histocompatibility complex (MHC). The transport of the antigenic peptides into the lumen of the endoplasmic reticulum (ER) and loading onto the MHC class I molecules is an essential process for the presentation to cytotoxic T lymphocytes. The delivery of these peptides is performed by the transporter associated with antigen processing (TAP). TAP is a heterodimer of TAP1 and TAP2, each subunit containing transmembrane domains and an ATP-binding motif. Sequence homology analysis revealed that TAP belongs to the superfamily of ATP-binding cassette transporters. Loss of TAP function leads to a loss of cell surface expression of MHC class I molecules. This may be a strategy for tumors and virus-infected cells to escape immune surveillance. Structure and function of the TAP complex as well as the implications of loss or downregulation of TAP is the topic of this review.     

Specificity of the proteasome and the TAP transporter.

The generation of antigenic peptides and their transport across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules are essential steps in antigen presentation to cytotoxic T lymphocytes. Recent studies have characterized the substrate specificities of the proteasome and the transporter associated with antigen processing. It is interesting to compare the specificity of this transporter to the wide spectrum of peptides generated by the proteasome, to the binding motifs of MHC class I molecules and in particular to the principles of T cell recognition.     

The ABC-transporter signature motif is required for peptide translocation but not peptide binding by TAP

The transporter associated with antigen processing (TAP) translocates peptides from their site of generation in the cytosol to the lumen of the endoplasmic reticulum for binding to MHC class I molecules. TAP is a member of the ATP-binding cassette (ABC) transporter family whose members utilize energy from ATP hydrolysis to translocate substrates across membranes. The highly conserved nucleotide-binding domains of ABC transporters couple ATP hydrolysis to substrate translocation by the membrane domains. The conserved 'signature motif' can be identified in the nucleotide-binding domains of all ABC transporters, and may play a role in ATP hydrolysis. Here we show that introduction of mutations into the signature motifs of either TAP1 or TAP2 inhibits the translocation of peptide without affecting binding of either peptide or ATP by TAP. We therefore conclude that the signature motifs in both TAP1 and TAP2 are required after peptide binding to facilitate peptide translocation by TAP.     

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ～ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.     

Intracellular peptide transporters in human – compartmentalization of the “peptidome”

In the human genome, the five adenosine triphosphate (ATP)-binding cassette (ABC) half transporters ABCB2 (TAP1), ABCB3 (TAP2), ABCB9 (TAP-like), and in part, also ABCB8 and ABCB10 are closely related with regard to their structural and functional properties. Although targeted to different cellular compartments such as the endoplasmic reticulum (ER), lysosomes, and mitochondria, they are involved in intracellular peptide trafficking across membranes. The transporter associated with antigen processing (TAP1 and TAP2) constitute a key machinery in the major histocompatibility complex (MHC) class I-mediated cellular immune defense against infected or malignantly transformed cells. TAP translocates the cellular "peptidome" derived primarily from cytosolic proteasomal degradation into the ER lumen for presentation by MHC class I molecules. The homodimeric ABCB9 (TAP-like) complex located in lysosomal compartments shares structural and functional similarities to TAP; however, its biological role seems to be different from the MHC I antigen processing. ABCB8 and ABCB10 are targeted to the inner mitochondrial membrane. MDL1, the yeast homologue of ABCB10, is involved in the export of peptides derived from proteolysis of inner-membrane proteins into the intermembrane space. As such peptides are presented as minor histocompatibility antigens on the surface of mammalian cells, a physiological role of ABCB10 in the antigen processing can be accounted.     

TAP genes and immunity

The transporter associated with antigen processing (TAP) is a member of the ATP-binding cassette transporter family that specializes in delivering cytosolic peptides to class I molecules in the endoplasmic reticulum. The TAP is a major target of genetic alteration in tumours and disruption by viral inhibitors. In some species, TAP genes have co-evolved with MHC class I molecules to deliver peptides that are customised for particular alleles. In humans, MHC class I polymorphism determines the level of tapasin-mediated association with TAP and subsequent peptide optimisation within the peptide-loading complex (PLC). MHC class I molecules that still load peptides without complexing to the TAP might be more resistant to viral interference of the PLC and less sensitive to competition for TAP by other class I allotypes.     

MHC class I assembly: out and about

The assembly of major histocompatibility complex (MHC) class I molecules with peptides is orchestrated by several assembly factors including the transporter associated with antigen processing (TAP) and tapasin, the endoplasmic reticulum (ER) oxido-reductases ERp57 and protein disulfide isomerase (PDI), the lectin chaperones calnexin and calreticulin, and the ER aminopeptidase (ERAAP). Typically, MHC class I molecules present endogenous antigens to cytotoxic T lymphocytes (CTLs). However, the initiation of CD8(+) T-cell responses against many pathogens and tumors also requires the presentation of exogenous antigens by MHC class I molecules. We discuss recent developments relating to interactions and mechanisms of function of the various assembly factors and pathways by which exogenous antigens access MHC class I molecules.     

Tapasin: an ER chaperone that controls MHC class I assembly with peptide

The stable assembly of MHC class I molecules with peptides in the endoplasmic reticulum (ER) involves several accessory molecules. One of these accessory molecules is tapasin, a transmembrane protein that tethers empty class I molecules to the peptide transporter associated with antigen processing (TAP). Here, evidence is presented that tapasin retains class I molecules in the ER until they acquire high-affinity peptides.     

Molecular mechanisms of class I major histocompatibility complex antigen processing and presentation

The presentation of antigenic peptides by class I major histocompatibility complex molecules plays a central role in the cellular immune response, since immune surveillance for detection of viral infections or malignant transformations is achieved by CD8+ T lymphocytes which inspect peptides, derived from intracellular proteins, bind to class I molecules on the surface of most cells. The transporter associated with antigen processing selectively translocates cytoplasmically derived peptides of appropriate sequence and length into the lumen of the endoplasmic reticulum where they associate with newly synthesized class I molecules. The translocated peptides are generated by multicatalytic and multisubunit proteasomes which degrade cytoplasmic proteins in a ATP-ubiquitin-dependent manner. This review discusses our current molecular understanding of class I antigen processing and presentation.     

The aminopeptidase ERAAP shapes the peptide repertoire displayed by major histocompatibility complex class I molecules

Major histocompatibility complex (MHC) class I molecules present thousands of peptides to allow CD8(+) T cells to detect abnormal intracellular proteins. The antigen-processing pathway for generating peptides begins in the cytoplasm, and the MHC molecules are loaded in the endoplasmic reticulum. However, the nature of peptide pool in the endoplasmic reticulum and the proteolytic events that occur in this compartment are unclear. We addressed these issues by generating mice lacking the endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP). We found that loss of ERAAP disrupted the generation of naturally processed peptides in the endoplasmic reticulum, decreased the stability of peptide-MHC class I complexes and diminished CD8(+) T cell responses. Thus, trimming of antigenic peptides by ERAAP in the endoplasmic reticulum is essential for the generation of the normal repertoire of processed peptides.     

TAP的研究进展

抗原处理相关转运体蛋白(transporter associated with antigen processing,TAP)在内源性抗原提呈过程中有重要作用,负责内源性抗原从胞浆到内质网(ER)腔的转运。TAP异二聚体由TAP1和TAP2蛋白组成,每个亚基各有一个核酸结合区(NBD)和一个跨膜区(TMD)。TAP对抗原肽的转运可分为不依赖ATP的TAP与抗原肽的结合和ATP依赖性的抗原肽到内质网的转运两个基本步骤。人类TAP1和TAP2等位基因在不同种族和地区有不同的分布。TAP与一些自身免疫性疾病、肿瘤和病毒感染性疾病的发生有一定的相关性。     

Interactions formed by individually expressed TAP1 and TAP2 polypeptide subunits

The transporter associated with antigen processing (TAP) supplies peptides into the lumen of the endoplasmic reticulum (ER) for binding by major histocompatibility complex (MHC) class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each a 'half-transporter' encoding a transmembrane domain and a nucleotide-binding domain. Immunoprecipitation of rat TAP1 and TAP2 expressed individually in the human TAP-deficient cell line, T2, revealed that both bound the endogenously expressed HLA-A2 and -B51 class I molecules. Using HLA-encoding recombinant vaccinia viruses HLA-A*2501, -B*2704, -B*3501 and -B*4402, alleles also associated with both TAP1 and TAP2. Thus, TAP1 and TAP2 do not appear to differ in their ability to interact with MHC class I alleles. Single TAP polypeptide subunits also formed MHC class I peptide-loading complexes, and their nucleotide-binding domains retained the ability to interact with ATP, and may permit the release of peptide-loaded MHC class I molecules in the absence of a peptide transport cycle. It is also demonstrated by chemical cross-linking that TAP2, but not TAP1, has the ability to form a homodimer complex both in whole cells and in detergent lysates. Together these data indicate that single TAP polypeptide subunits possess many of the features of the TAP heterodimer, demonstrating them to be useful models in the study of ATP-binding cassette (ABC) transporters.     

TAP-translocated peptides specifically bind proteins in the endoplasmic reticulum,including gp96, protein disulfide isomerase and calreticulin

The endoplasmic reticulum (ER) membrane-embedded transporter associated with antigen processing (TAP) associates with peptides in the cytosol and translocates these into the ER lumen. Here, MHC class I molecules bind a subset of these peptides and the remainder is either removed or degraded, or may be retained in the ER in association with other proteins. We have visualized peptide-binding proteins in the ER using radioactive peptides with a photoreactive group. Besides TAP, two proteins were identified as gp96 and protein disulfide isomerase (PDI). Calreticulin, previously found in complex with TAP, only binds glycosylated peptides. In addition, two as yet unidentified, ER luminal glycoproteins (gp120 and gp170) were visualized. The effects of peptide size and sequence on binding to the ER-resident proteins were studied by using partially degenerated peptides with photoreactive side chains. All identified proteins were able to bind peptides within the size range of peptides translocated by TAP, from 8 to more than 20 amino acids. Whereas PDI associated with all peptides tested, gp96 and gp120 showed a clear sequence preference for non-charged amino acids at positions 2 and 9 in 9mer peptides. Thus various ER proteins, other than the MHC class I heterodimer and TAP, are able to interact with peptides albeit with a different substrate selectivity.     

Major histocompatibility complex class I-restricted antigen processing and presentation

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8-expressing cytotoxic T lymphocytes (CTLs). This antigen recognition system is critically important for immune surveillance against viruses and tumors. Most class I-binding peptides are generated in the cytosol, as side products from the degradation of misfolded proteins by proteasomes. A subset of the resulting peptides are translocated across the endoplasmic reticulum (ER) membrane by a dedicated peptide transporter, and these peptides are then loaded onto peptide-receptive class I molecules in the ER. The stable assembly of class I molecules with peptides is controlled by a variety of accessory proteins, including chaperones with general housekeeping functions and factors with dedicated roles in class I assembly. Peptide-filled class I molecules are then delivered to the cell surface for recognition by CTLs. This highly regulated process permits the host to rapidly counter invading pathogens with strong and sustained CTL responses and, at the same time, avoid misguided attacks. Here, how the class I antigen processing machinery accomplishes this daunting task is reviewed.     

Heat shock protein 70 moderately enhances peptide binding and transport by the transporter associated with antigen processing

Hsp70 molecules are capable of binding antigenic peptides and eliciting CTL responses to the bound peptide. However, the precise mechanism for the induction of CTL has not been determined. One possibility is that hsp molecules can directly shuttle peptides in the MHC class I antigen processing and presentation pathway, as previously postulated. Here, we have addressed this issue by testing the effect of purified hsp70 molecules on peptide binding and transport by the transporter associated with antigen processing (TAP). Our results indicate that purified hsp70 molecules moderately enhance TAP function. In addition, we detect a physical association between hsp70 molecules and TAP, as well as the homologous drug transporter P-glycoprotein. We conclude that while hsp70 molecules may not be directly involved in the delivery of peptide to TAP, they may play an important role in TAP transport by binding to TAP and promoting its function.     

Proteolysis and class I major histocompatibility complex antigen presentation

Summary: The class I major histocompatibility complex (MHC class 1) presents 8–10 residue peptides to cytotoxic T lymphocytes. Most of these antigenic peptides are generated during protein degradation in the cytoplasm and are then transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP), Several lines of evidence have indicated that the proteasome is the major proteolytic activity responsible for generation of antigenic peptides—probably most conclusive has been the finding that specific inhibitors of die proteasome block antigen presentation. However, other proteases (e.g. the signal peptidase) may also generate some epitopes, particularly those on certain MHC class I alleles. The proteasome is responsible for generating the precise C termini of many presented peptides, and appears to be the only activity in ceils that can make this cleavage. In contrast, aminopeptidases in the cytoplasm and endoplasmic reticulum can trim the N terminus of extended peptides to their proper size. Interestingly, the cellular content of proteases involved in the production and destruction of antigenic peptides is modified by inter-feron-γ (IFN-γ) treatment of cells, IFN-γ indicates the expression of three new proteasome β submits that are preferentially incorporated into new proteasomes and alter their pattern of peptidase activities. These changes are likely to enhance the yield of peptides with C termini appropriate for MHC binding and have been shown to enhance the presentation of at least some antigens. IFN-γ also upregulates leucine aminopeptidase, which should promote the removal of N-terminal flanking residues of antigenic peptides. Also, this cytokine downregulates the expression of a metallo-proteinase, thimet oligopeptidase that actively destroys many antigenic peptides. Thus, IFN-γ appears to increase the supply of peptides by stimulating their generation and decreasing their destruction. The specificity and content of these various proteases should determine the amount of peptides available for antigen presentation. Also, the efficiency with which a peptide is presented is determined by the protein's half life (e.g. its ubiquitination rate) and the sequences flanking antigenic peptides, which influence the rates of proteolytic cleavage and destruction.     

Characterization and allelic variation of the transporters associated with antigen processing (TAP) genes in the domestic dog (Canis lupus familiaris)

The function of the transporters associated with antigen processing (TAP) complex is to shuttle antigenic peptides from the cytosol to the endoplasmic reticulum to load MHC class I molecules for CD8⁺ T-cell immunosurveillance. Here we report the promoter and coding regions of the canine TAP1 and TAP2 genes, which encode the homologous subunits forming the TAP heterodimer. By sampling genetically divergent breeds, polymorphisms in both genes were identified, although there were few amino acid differences between alleles. Splice variants were also found. When aligned to TAP genes of other species, functional regions appeared conserved, and upon phylogenetic analysis, canine sequences segregated appropriately with their orthologs. Transfer of the canine TAP2 gene into a murine TAP2-defective cell line rescued surface MHC class I expression, confirming exporter function. This data should prove useful in investigating the association of specific TAP defects or alleles with immunity to intracellular pathogens and cancer in dogs.     

How does TAP associate with MHC class I molecules?

Major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER) are in physical association with a number of cofactors, including the transporter associated with antigen processing (TAP) and a calcium-binding chaperone. Here, Tam Ellioti suggests a molecular model for the way in which these cofactors could regulate the assembly and release of newly synthesized MHC class I molecules.     

Accessory proteins and the assembly of human class I MHC molecules: a molecular and structural perspective

The cell-surface presentation of antigenic peptides by class I major histocompatibility complex (MHC) molecules to CD8+ T-cell receptors is part of an immune surveillance mechanism aimed at detecting foreign antigens. This process is initiated in the endoplasmic reticulum (ER) with the folding and assembly of class I MHC molecules which are then transported to the cell surface via the secretory pathway. In recent years, several accessory proteins have been identified as key components of the class I maturation process in the ER. These proteins include the lectin chaperones calnexin (CNX) and calreticulin (CRT), the thiol-dependent oxidoreductase ERp57, the transporter associated with antigen processing (TAP), and the protein tapasin. This review presents the most recent advances made in characterizing the biochemical and structural properties of these proteins, and discusses how this knowledge advances our current understanding of the molecular events underlying the folding and assembly of human class I MHC molecules in the ER.     

ERAAP synergizes with MHC class I molecules to make the final cut in the antigenic peptide precursors in the endoplasmic reticulum

The major histocompatibility complex class I molecules display peptides (pMHC I) on the cell surface for immune surveillance by CD8(+) T cells. These peptides are generated by proteolysis of intracellular polypeptides by the proteasome in the cytoplasm and then in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with antigen processing (ERAAP). To define the unknown mechanism of ERAAP function in vivo, we analyzed naturally processed peptides in cells with or without appropriate MHC I and ERAAP. In the absence of MHC I, ERAAP degraded the antigenic precursors in the ER. However, MHC I molecules could bind proteolytic intermediates and were essential for generation of the final peptide by ERAAP. Thus, ERAAP synergizes with MHC I to generate the final pMHC I repertoire.